Method of preparation of an oxidized derivative of hyaluronic acid and a method of modification thereof

ABSTRACT

The invention relates to a new method of preparation of a hyaluronan derivative with an aldehydic group in the position (6) of the polysaccharide glucosamine part. The hyaluronic acid oxidation can be performed by means of TEMPO/NaCIO or TEMPO/TCC systems in a protic environment with or without the presence of anorganic salts. Thus prepared aldehyde can be used for binding amines, diamines, amino acids, peptides and other compounds containing an amino group, e.g. by means of the reductive amination with NaBH 3 CN in water or in a water-organic solvent system. When a diamine or compounds containing three or more amino groups are used, cross-linked hyaluronan derivatives can be prepared. Cross-linked derivatives can be also prepared by a reaction of the aldehyde with a hyaluronan substituted by an amino-alkyl group HA-alkyl-NH 2 .

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of PCT/CZ2010/000129, filedDec. 10, 2010, which claims priority to Czech Republic application No.PV 2009-835, filed Dec. 11, 2009, the disclosures of which are herebyincorporated by reference herein. This application is also related toPCT/CZ2010/000128, filed Dec. 10, 2010.

FIELD OF THE INVENTION

The present invention relates to a new method of preparation of ahyaluronic acid derivative containing an aldehydic group —CH═O insteadof the primary hydroxyl group —CH₂—OH. The oxidation can be performed inprotic solvents by use of a system of 4-substituted-TEMPO(2,2,6,6-tetramethyl-piperidinyloxyl)/co-oxidant:

wherein R is hydrogen or any substitute and the co-oxidant is sodiumhypochlorite (NaClO) or trichloroisocyanuric acid (TCC).

BACKGROUND OF THE INVENTION

The hyaluronic acid is an important polysaccharide, composed of tworepeating units of β-(1,3)-D-glucuronic acid andβ-(1,4)-N-acetyl-D-glucosamine. The molecular weight, depending on themethod of isolation and on the source material, is within the range from5·10⁴ to 5·10⁶ g·mol⁻¹. The hyaluronic acid or its sodium salthyaluronan is an essential component of connective tissues, synovialfluid of joints, and plays an important role in biological processes,such as hydratation, organization of proteoglycans, celldifferentiation, proliferation and angiogenesis. The hyaluronic acid isa considerably hydrophilic polysaccharide and is soluble in water in theform of a salt in the whole range of pH.

Oxidation of the Hyaluronic Acid

Oxidation of polysaccharides is a process in which the oxidation degreeof the polysaccharide functional groups is changing. Most frequently,carboxylic acids or aldehydes are formed, which can dramatically changethe properties of the polysaccharide. In most cases, the reaction isperformed by use of agents containing atoms in higher oxidation degrees.

The method of selective oxidation of saccharides on the primary hydroxylgroup, described in Angelino, European Journal of Organic Chemistry2006, 19, 4323-4326, the system of 2,2,6,6-tetramethyl-1-piperidinyloxylradical TEMPO/TCC in DMF at the temperature of 0° C. was used, with thecorresponding aldehyde as the main product.

TEMPO is a relatively stable radical which can exist in reaction inthree redox forms. Only the oxidized form of TEMPO is effective foralcohols or geminal diols oxidation.

A catalytic amount of TEMPO is added to the reaction and therefore, itis necessary to use a co-oxidant which restores the presence of theTEMPO oxidized form.

2,2,6,6-tetramethyl-1-piperidinyloxyl radical (TEMPO)- andNaOCl-mediated oxidation of the primary hydroxyl group of hyaluronan toa carboxylic acid was performed at pH 10.2 and at the temperature of 0°C. (Scheme 2) (Carbohydr Res 2000, 327 (4), 455-61).

Analogous to other polysaccharides, a high regioselectivity and slightdegradation of the polymer were observed. An increase of theconcentration of the salt (NaBr, NaCl, Na₂SO₄) in the solution caused adecrease in the oxidation rate.

Oxidation of hyaluronan by use of TEMPO/NaClO system was described inthe patent application WO 02/18448 A2. The authors also dealt withinteractions of percarboxylated polysaccharides, while formingbiological complexes.

The rate of oxidation of HA and other polysaccharides by use of sodiumperiodate was studied by Scott et al. (Scheme 3) (Histochemie 1969, 19(2), 155-61).

The factors such as the chain length, substitution, polymerconfiguration and temperature were studied and quantified. The use ofNaIO₄ for an oxidation of hyaluronan was also disclosed in the U.S. Pat.No. 6,683,064 and U.S. Pat. No. 6,953,784.

Model reactions of HA analogues with low molecular weight in aphysiological buffer were studied (Carbohydr Res 1999, 321, (3-4),228-34). Oxidation products of the glucuronic and glucosamine parts wereidentified by GC-MS analysis. The results also suggest that theoxidation is performed primarily on the glucuronic part, while themeso-tartaric acid is the main product and may be used as a biomarker ofthe hyaluronan oxidation.

Use of an Oxidized HA in Cross-Linking Reactions

The use of an oxidized HA for the preparation of cross-linked hydrogelswas described by Weng et al. (Scheme 4), J Biomed Mater Res A 2008, 85(2), 352-65. Two precursors were used in this case: a partially oxidizedhyaluronan and gelatin:

The physico-chemical properties of the resulting hydrogels have beenelucidated by instrumental analyses FT-IR, SEM (scanning electronMicroscopy) and rheometry. Increasing the oxidation degree of thehyaluronan lead to a corresponding increase of hydrogels compatibilityand decrease of water absorption capacity. Dermal fibroblasts were usedto study the cell-hydrogel interactions. Both the hydrogels and theirdegradation products are biocompatible, as proved by the long-term cellviability assay. When cultured with cells, the hydrogel underwent adegradation within 4 weeks, with an obvious loss of cohesiveness. Thegood biocompatibility and biodegradability was further demonstrated inmice subdermal implantations. Lastly, in vitro and in vivo depositionsof extracellular matrix in hydrogels were demonstrated by SEM analysis.

The method of preparation of cross-linked HA from an oxidized hyaluronanand gelatin by a water-in-oil-emulsion method, where a 3-dimensionalhydrogel is formed in the absence of any external cross-linker, wasdescribed in the publication of Weng et al., Biomaterials 2008, 29,(31), 4149-56. In this work, incorporation of model drugs into thehydrogel structure (encapsulation) and their releasing throughmacrophages were studied by HPLC.

The preparation of elastic hydro gels by coupling the HA oxidized toHA-aldehyde by means of sodium periodate and the HA modified with adipicacid dihydrazide, was described by Sahiner et al., (Scheme 5), J.Biomater. Sci. Polym. Ed 2008, 19 (2), 223-43.

The resulting derivatives did not have any observable effect on theproliferation of the cultured fibroblasts, as shown by a MTT assay.

SUMMARY OF THE INVENTION

The present invention relates to an improvement of a method of selectiveoxidation of the primary hydroxyl group of the hyaluronic acid in theposition 6 of the polysaccharide glucosamine part to aldehyde. The newreaction is performed using a system of2,2,6,6-tetramethyl-1-piperidinyloxyl radical TEMPO/co-oxidant.Approaches published up to date have introduced either an aldehydicgroup to the position 2 and 3 of the hyaluronan glucuronic part, whileopening the saccharide ring (scheme 6, structure 2), or a carboxyl groupto the position 6 of the hyaluronan glucosamine part (scheme 6,structure 1).

The method according to the invention is more advantageous in that therespective oxidation product (structure 3, scheme 6) maintains thestructure of conjugated saccharide rings. Ring opening in the productoxidized to dialdehyde (structure 2, scheme 6) gives rise to the chainlinearity “breakage” and, therefore, a significant change of thepolysaccharide 3-dimensional structure compared to the non-modifiedhyaluronan. Although in the product oxidized to a carboxylic acid(structure 1, scheme 6) the chain linearity “breakage” does not occur,the carboxyl group does not enable such various modification (binding)possibilities like the aldehydic group. As the carboxyl group is acomponent of the non-modified polysaccharide already, the oxidation tothe structure 1 (scheme 6) brings only an increase of the polysaccharidepolarity, not a development of new quality utilizable for binding newsubstitutes.

It is known that an aldehydic group with a bound alkyl group exists inwater in the so-called geminal diol form HA-CH(OH)₂, which reacts withnucleophiles similarly as aldehydes do. In aqueous solutions, more than95% of the hyaluronan oxidized in the position 6 of the glucosamine part(product 3, scheme 6) exists in the form of a geminal diol, asdemonstrated by NMR spectroscopy.

In method according to the present invention, the hyaluronic acid isdissolved in water, then an oxidizing agent is added and the mixture isstirred at the temperature of −10 to 20° C., preferably at 0° C., for 1to 150 hours, preferably for 24 hours.

The prepared oxidized hyaluronan can be used for binding compoundscontaining for example an amino group. The binding can be realizedeither in an imine form or after the reduction in an amine form(reductive amination) (scheme 7):

Both degrees of this modification are performed in an aqueous solution,the reduction is performed by means of NaBH₃CN. Both degrees of thereaction described in scheme 7 can be performed in one step.

The modification of the hyaluronic acid derivative can be performed by areaction of the oxidized derivative with an amine of the general formulaH₂N—R or with a hyaluronan substituted by an —R—NH₂ group, wherein R isan alkyl, linear or branched chain C1-C30, optionally containingaromatic or heteroaromatic groups. This amine can be an alkylamine, e.g.butylamine or hexanediamine, amino acid, peptide or polysaccharidecontaining a free amino group. In case of using a diamine or compoundscontaining three or more amino groups, cross-linked hyaluronanderivatives can be prepared. The cross-linked derivatives can beprepared also by the reaction of an aldehyde with a hyaluronansubstituted by an aminoalkyl group HA-alkyl-NH₂.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

DS=substitution degree=100%*the molar amount of the bound substitute/themolar amount of all the polysaccharide dimers.

The term “equivalent (eq)” as used herein, refers to a hyaluronic aciddimer, unless otherwise indicated. The percentages as used herein mean apercentage by weight, unless otherwise indicated.

The molecular weight of the starting hyaluronan (source: CPN spol. sr.o., Dolní Dobrou{hacek over (c)}, CZ) was determined by the SEC-MALLSassay.

Example 1 Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System

An aqueous solution of NaClO (0.5 eq) was gradually added to an aqueoussolution of hyaluronan (1%) (0.1 g, 20 kDa) (containing 10% by weight ofNaCl), TEMPO (0.01 eq) and NaHCO₃ (30 eq). The mixture was stirred for24 hours at the temperature of −5° C. The solution was then diluted to0.2% with distilled water and dialyzed against the mixture (0.1% NaCl,0.1% NaHCO₃) 3×5 liters (once per day) and against distilled water 7×5liters (twice per day). The resulting solution was evaporated andanalyzed.

DS 10% (determined by NMR)

¹H NMR (D₂O) δ5.26 (s, 1H, polymer-CH(OH)₂)

HSQC (D₂O) cross signal 5.26 ppm (¹H)-90 ppm (¹³C) (polymer-CH(OH)₂)

FT-IR (KBr) 1740 cm⁻¹ —CH═O

Example 2 Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System

An aqueous solution of NaClO (0.5 eq) was gradually added to an aqueoussolution of hyaluronan (0.5%) (0.1 g, 2000 kDa) (containing 10% byweight of NaCl), TEMPO (0.01 eq) and NaHCO₃ (30 eq). The mixture wasstirred for 24 hours at the temperature of −5° C. The solution was thendiluted to 0.2% with distilled water and dialyzed against the mixture(0.1% NaCl, 0.1% NaHCO₃) 3×5 liters (once per day) and against distilledwater 7×5 liters (twice per day). The resulting solution was evaporatedand analyzed.

DS 10% (determined by NMR, for details see Example 1)

Example 3 Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System

An aqueous solution of NaClO (0.5 eq) was gradually added to an aqueoussolution of hyaluronan (1%) (0.1 g, 20 kDa) (containing 10% by weight ofNaCl), 4-acetamido-TEMPO (0.01 eq) and NaHCO₃ (30 eq). The mixture wasstirred for 24 hours at the temperature of −5° C. The solution was thendiluted to 0.2% with distilled water and dialyzed against the mixture(0.1% NaCl, 0.1% NaHCO₃) 3×5 liters (once per day) and against distilledwater 7×5 liters (twice per day). The resulting solution was evaporatedand analyzed.

DS 10% (determined by NMR, for details see Example 1)

Example 4 Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System

An aqueous solution of NaCIO (0.5 eq) was gradually added to an aqueoussolution of hyaluronan (1%) (0.1 g, 20 kDa) (containing 10% by weight ofNaCl), 4-acetamido-TEMPO (0.05 eq) and NaHCO₃ (30 eq). The mixture wasstirred for 10 hours at the temperature of −10° C. The solution was thendiluted to 0.2% with distilled water and dialyzed against the mixture(0.1% NaCl, 0.1% NaHCO₃) 3×5 liters (once per day) and against distilledwater 7×5 liters (twice per day). The resulting solution was evaporatedand analyzed.

DS 10% (determined by NMR, for details see Example 1)

Example 5 Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System

An aqueous solution of NaClO (0.3 eq) was gradually added to an aqueoussolution of hyaluronan (1%) (0.1 g, 20 kDa), 4-acetamido-TEMPO (0.1 eq)and NaHCO₃ (30 eq). The mixture was stirred for 1 hour at thetemperature of 20° C. The solution was then diluted to 0.2% withdistilled water and dialyzed against the mixture (0.1% NaCl, 0.1%NaHCO₃) 3×5 liters (once per day) and against distilled water 7×5 liters(twice per day). The resulting solution was evaporated and analyzed.

DS 5% (determined by NMR, for details see Example 1)

Example 6 Oxidation of the Hyaluronic Acid by the TEMPO/TCC System

4-acetamido-TEMPO (0.01 eq) and TCC (0.2 eq) were added to a solution ofhyaluronic acid (1%) (0.1 g, 20 kDa) in the mixture ofN,N-dimethylformamide/water 70/30. The mixture was stirred for 24 hoursat the temperature of −5° C. The solution was then diluted to 0.1% anddialyzed against the mixture (0.1% NaCl, 0.1% NaHCO₃) 3×5 liters (onceper day) and against distilled water 7×5 liters (twice per day). Theresulting solution was evaporated and analyzed.

DS 5% (determined by NMR, for details see Example 1)

Example 7 Oxidation of the Hyaluronic Acid by the TEMPO/TCC System

4-acetamido-TEMPO (0.01 eq) and TCC (0.2 eq) were added to a solution ofhyaluronic acid (1%) (0.1 g, 20 kDa) in the mixture ofN,N-dimethylformamide/water 90/10. The mixture was stirred for 24 hoursat the temperature of 20° C. The solution was then diluted to 0.1% anddialyzed against the mixture (0.1% NaCl, 0.1% NaHCO₃) 3×5 liters (onceper day) and against distilled water 7×5 liters (twice per day). Theresulting solution was evaporated and analyzed.

DS 10% (determined by NMR, for details see Example 1)

Example 8 Oxidation of the Hyaluronic Acid by the TEMPO/TCC System

4-acetamido-TEMPO (0.01 eq) and TCC (1 eq) were added to a solution ofhyaluronic acid (1%) (0.1 g, 20 kDa) in the mixture ofN,N-dimethylformamide/water 40/60. The mixture was stirred for 24 hoursat the temperature of −5° C. The solution was then diluted to 0.1% anddialyzed against the mixture (0.1% NaCl, 0.1% NaHCO₃) 3×5 liters (onceper day) and against distilled water 7×5 liters (twice per day). Theresulting solution was evaporated and analyzed.

DS 15% (determined by NMR, for details see Example 1)

Example 9 Oxidation of the Hyaluronic Acid by the TEMPO/TCC System

4-acetamido-TEMPO (0.01 eq) and TCC (1 eq) were added to a solution ofhyaluronic acid (0.5%) (0.1 g, 2000 kDa) in the mixture ofN,N-dimethylformamide/water 40/60. The mixture was stirred for 24 hoursat the temperature of −5° C. The solution was then diluted to 0.1% anddialyzed against the mixture (0.1% NaCl, 0.1% NaHCO₃) 3×5 liters (onceper day) and against distilled water 7×5 liters (twice per day). Theresulting solution was evaporated and analyzed.

DS 15% (determined by NMR, for details see Example 1)

Example 10 Oxidation of the Hyaluronic Acid by the TEMPO/TCC System

TEMPO (0.01 eq) and TCC (0.2 eq) were added to a solution of hyaluronicacid (1%) (0.1 g, 20 kDa) in the mixture of N,N-dimethylformamide/water70/30. The mixture was stirred for 1 hour at the temperature of 20° C.The solution was then diluted to 0.1% and dialyzed against the mixture(0.1% NaCl, 0.1% NaHCO₃) 3×5 liters (once per day) and against distilledwater 7×5 liters (twice per day). The resulting solution was evaporatedand analyzed.

DS 5% (determined by NMR, for details see Example 1)

Example 11 Oxidation of the Hyaluronic Acid by the TEMPO/TCC System

TEMPO (0.01 eq) and TCC (0.2 eq) were added to a hyaluronic acidsolution (1%) (0.1 g, 20 kDa) in the mixture of DMF/water 70/30. Themixture was stirred for 100 hours at the temperature of −5° C. Thesolution was then diluted to 0.1% and dialyzed against the mixture (0.1%NaCl, 0.1% NaHCO₃) 3×5 liters (once per day) and against distilled water7×5 liters (twice per day). The resulting solution was evaporated andanalyzed.

DS (determined by NMR, for details see Example 1)

Example 12 Oxidation of the Hyaluronic Acid by the TEMPO/NaBrO System

An aqueous solution of NaClO (0.5 eq) was gradually added to an aqueoussolution of hyaluronan (1%) (0.1 g, 20 kDa) (containing 5% by weight ofNaBr), 4-acetamido-TEMPO (0.5 eq) and NaHCO₃ (30 eq). The mixture wasstirred for 24 hours at the temperature of 0° C. The solution was thendiluted to 0.2% with distilled water and dialyzed against the mixture(0.1% NaClO, 0.1% NaHCO₃) 3×5 liters (once per day) and againstdistilled water 7×5 liters (twice per day). The resulting solution wasevaporated and analyzed.

DS 14% (determined by NMR, for details see Example 1)

Example 13 Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System

An aqueous solution of NaClO (0.5 eq) was gradually added to an aqueoussolution of hyaluronan (1%) (0.1 g, 200 kDa) (containing 10% by weightof NaBr), TEMPO (0.5 eq) and NaHCO₃ (5 eq). The mixture was stirred for24 hours at the temperature of 0° C. The solution was then diluted to0.2% with distilled water and dialyzed against mixture (0.1% NaCl, 0.1%NaHCO₃) 3×5 liters (once per day) and against distilled water 7×5 liters(twice per day). The resulting solution was evaporated and analyzed.

DS 18% (determined by NMR, for details see Example 1)

Example 14 Reaction of the Oxidized Hyaluronan with an Amine

Butylamine (0.4 eq) was added to an aqueous solution of the oxidizedhyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). Themixture was stirred for 24 hours at the temperature of 20° C. Thesolution was then precipitated with a double amount of acetone and 0.1ml of a saturated aqueous solution of NaCl, filtrated and dried invacuum. The resulting yellow material was then analyzed.

UV-VIS 328 nm, n→π*transition —CH═N—

Example 15 Reaction of Oxidized Hyaluronan with Butylamine andSubsequent Reduction

Butylamine (0.4 eq) was added to an aqueous solution of the oxidizedhyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). Themixture was stirred for 24 hours at the temperature of 20° C. Thesolution was then mixed with NaBH₃CN in 0.5 ml of water (3 eq). Themixture was stirred for 24 hours at the temperature of 20° C. Thesolution was then diluted to 0.1% and dialyzed against the mixture (0.1%NaCl, 0.1% NaHCO₃) 3×5 liters (once per day) and against distilled water7×5 liters (twice per day). The resulting solution was evaporated andanalyzed.

DS 15% (determined by NMR)

¹H NMR (D₂O) δ3.05 (m, 2H, polymer-CH₂—NH—CH₂—), 1.60 (m, 2H,polymer-CH₂—NH—CH₂—CH₂—), 1.35 (m, 2H, polymer-CH₂—NH—CH₂—CH₂—CH₂—),0.85 (m, 3H, —CH₂—CH₃)

DOSY NMR (D₂O) log D (0.85 ppm, —CH₂—CH₃)˜−10.3 m²/s

-   -   log D (1.35 ppm, polymer-CH₂—NH—CH₂—CH₂—CH₂—)˜−10.3 m²/s    -   log D (1.60 ppm, polymer-CH₂—NH—CH₂—CH₂—)˜−10.3 m²/s    -   log D (3.05 ppm, polymer-CH₂—NH—CH₂—)˜−10.3 m²/s    -   log D (2.03 ppm, CH₃—CO—NH-polymer)˜−10.3 m²/s    -   log D (H₂O)˜−8.6 m²/s

Example 16 Reaction of the Oxidized Hyaluronan with a Diamine and theSubsequent Reduction

Hexanediamine (0.4 eq) was added to an aqueous solution of the oxidizedhyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). Themixture was stirred for 24 hours at the temperature of 20° C. Thesolution was then mixed with NaBH₃CN (3 eq) in 0.5 ml of water. Themixture was stirred for 24 hours at the temperature of 20° C. Thesolution was then diluted to 0.1% and dialyzed against the mixture (0.1%NaCl, 0.1% NaHCO₃) 3×5 liters (once per day) and against distilled water7×5 liters (twice per day). The resulting solution was evaporated andanalyzed.

DS 16% (determined by NMR)

¹H NMR (D₂O) δ3.12 (m, 2H, polymer-CH₂—NH—CH₂—), 3.02 (m, 2H, —CH₂—NH₂),1.7 (m, 4H, —NH—CH₂—CH₂—CH₂—CH₂—CH₂—), 1.45 (m, 411,—NH—CH₂—CH₂—CH₂—CH₂—CH₂—)

DOSY (D₂O) log D (1.45 ppm, —NH—CH₂—CH₂—CH₂—CH₂—CH₂—)˜−10.5 m²/s

-   -   log D (1.7 ppm, —NH—CH₂—CH₂—CH₂—CH₂—CH₂—)˜−10.5 m²/s    -   log D (3.02 ppm, —CH₂—NH₂)˜−10.5 m²/s    -   log D (2.03 ppm, CH₃—CO—NH-polymer)˜−10.5 m²/s    -   log D (H₂O)˜−8.7 m²/s

Example 17 Reaction of the Oxidized Hyaluronan with an Amine-Hyaluronan

A 1% aqueous solution of hyaluronan derivative substituted byhexanediamine (1 eq, DS=16%, Example 16) was added to an aqueoussolution of oxidized hyaluronic acid (1%) (0.1 g, oxidation degreeDS=18%, Example 13) at the temperature of 20° C. The resulting insolublecompact gummy precipitate, obtained after several minutes, wasmechanically ground to small pieces, filtrated and dried under reducedpressure.

FT-IR (KBr) 1700 cm⁻¹

Example 18 Reductive Amination of the Oxidized Hyaluronan with Lysine

Lysine (0.3 eq) was added to an aqueous solution of oxidized hyaluronicacid (1%) (0.1 g, oxidation degree DS=18%, Example 13). The mixture wasstirred for 24 hours at the temperature of 20° C. The solution was thenmixed with NaBH₃CN (3 eq) in 0.5 ml of water. The mixture was stirredfor 24 hours at the temperature of 20° C. The solution was diluted to0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO₃) 3×5liters (once per day) and against distilled water 7×5 liters (twice perday). The resulting solution was evaporated and analyzed.

DS 14% (determined by NMR)

¹H, HSQC, DOSY NMR (2% NaOD/D₂O): δ1.33 (m, 2H, —CH—CH₂—CH₂—), 1.48 (m,2H, —CH—CH₂—CH₂—CH₂—), 1.55 (m, 1H, —CH—CHH—), 1.63 (m, 1H, —CH—CHH—),2.62 (m, 2H, —CH—CH₂—CH₂—CH₂—CH₂—), 2.65 (m, 1H, polymer-CHH—NH—), 2.99(m, 1H, polymer-CHH—NH—), 3.16 (m, 1H, —CH—CH₂—).

Example 19 Reductive Amination of the Oxidized Hyaluronan with Lysine

Lysine (20 eq) was added to an aqueous solution of the oxidizedhyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). Themixture was stirred for 24 hours at the temperature of 20° C. Thesolution was then mixed with NaBH₃CN (10 eq) in 0.5 ml of water. Themixture was stirred for 24 hours at the temperature of 20° C. Thesolution was diluted to 0.1% and dialyzed against the mixture (0.1%NaCl, 0.1% NaHCO₃) 3×5 liters (once per day) and against distilled water7×5 liters (twice per day). The resulting solution was evaporated andanalyzed.

DS 17% (determined by NMR, Example 10)

Example 20 Reductive Amination of the Oxidized Hyaluronan with Serine

Serine (0.3 eq) was added to an aqueous solution of the oxidizedhyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). Themixture was stirred for 1 min at the temperature of 20° C. The solutionwas then mixed with NaBH₃CN (3 eq) in 0.5 ml of water.

The mixture was stirred for 24 hours at the temperature of 20° C. Thesolution was diluted to 0.1% and dialyzed against the mixture (0.1%NaCl, 0.1% NaHCO₃) 3×5 liters (once per day) and against distilled water7×5 liters (twice per day). The resulting solution was evaporated andanalyzed.

DS 14% (determined by NMR)

¹H, HSQC, DOSY NMR (2% NaOD/D₂O): δ2.74 (m, 1H, polymer-CHH—NH—), 3.08(m, 1H, polymer-CHH—NH—), 3.21 (m, 1H, —CH—CH₂—OH), 3.72 (m, 1H,—CH—CHH—OH), 3.78 (m, 1H, —CH—CHH—OH).

Example 21 Reductive Amination of the Oxidized Hyaluronan with Arginine

Arginine (0.3 eq) was added to an aqueous solution of the oxidizedhyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). Themixture was stirred for 100 hours at the temperature of 20° C. Thesolution was then mixed with NaBH₃CN (3 eq) in 0.5 ml of water. Themixture was stirred for 24 hours at the temperature of 20° C. Thesolution was diluted to 0.1% and dialyzed against the mixture (0.1%NaCl, 0.1% NaHCO₃) 3×5 liters (once per day) and against distilled water7×5 liters (twice per day). The resulting solution was evaporated andanalyzed.

DS 11% (determined by NMR)

¹H, HSQC, DOSY NMR (2% NaOD/D₂O): δ1.61 (m, 2H, —CH—CH₂—CH₂—), 1.63 (m,1H, —CH—CHH—), 1.70 (m, 1H, —CH—CHH—), 2.65 (m, 1H, polymer-CHH—NH—),3.01 (m, 1H, polymer-CHH—NH—), 3.13 (m, 1H, —CH—CH₂—), 3.21 (m, 2H,—CH—CH₂—CH₂—CH₂—).

Example 22 Reductive Amination of the Oxidized Hyaluronan withPentapeptide PAL-KTTKS (palmytoyl-Lys-Thr-Thr-Lys-Ser)

1% solution of substituted pentapeptide PAL-KTTKS (0.05 eq) in isopropylalcohol was added to a solution of the oxidized hyaluronic acid (1%)(0.1 g, oxidation degree DS=18%, Example 13) in the system ofwater/isopropanol 2/1. The mixture was stirred for 24 hours at thetemperature of 20° C. The solution was then mixed with NaBH₃CN in 0.5 mlof water (3 eq). The mixture was stirred for 24 hours at the temperatureof 20° C. The resulting solution was diluted to 0.1% and dialyzedagainst the mixture (0.1% NaCl, 0.1% NaHCO₃) 3×5 liters (once per day)and against distilled water 7×5 liters (twice per day). The resultingsolution was then evaporated and analyzed.

DS 9% (determined by NMR)

¹H, HSQC, DOSP NMR (2% NaOD/D₂O): δ1.61 (m, 2H, —CH—CH₂—CH₂—), 1.63 (m,1H, —CH—CHH—), 1.70 (m, 1H, —CH—CHH—), 2.65 (m, 1H, polymer-CHH—NH—),3.01 (m, 1H, polymer-CHH—NH—), 3.13 (m, 1H, —CH—CH₂—), 3.21 (m, 2H,—CH—CH₂—CH₂—CH₂—).

The invention claimed is:
 1. A method of modification of a hyaluronicacid derivative comprising: reacting hyaluronic acid with 4-R-TEMPO anda co-oxidant in a protic solvent, where R is a linear or branched alkylchain, optionally containing aromatic or heteroaromatic groups, whereinthe oxidized derivative of hyaluronic acid is selectively oxidized to analdehyde at position 6 of the glucosamine portion of the derivative and4-R-TEMPO is present in an amount within the range from 0.005 to 0.5molar equivalent with respect to the hyaluronic acid; and reacting theoxidized hyaluronic acid derivative with either a compound of a generalformula H₂N-R, where the general formula H₂N-R defines an amino acid,peptide, or polysaccharide containing a free amino group or where R is alinear or branched C₁—C₃₀ alkyl chain, optionally containing aromatic orheteroaromatic groups, thereby defining an amine, or with a hyaluronan,substituted by an -R-NH₂ group, where R is a linear or branched C₁—C₃₀alkyl chain, optionally containing aromatic or heteroaromatic groups, toform an imino form of HA of a general formula I


2. The method according to claim 1, wherein the co-oxidant includessodium hypochlorite or TCC (trichlorocyanuric acid).
 3. The methodaccording to claim 1, wherein the hyaluronic acid is in the form of afree acid or a salt.
 4. The method according to claim 1, wherein thehyaluronic acid molecular weight is within the range from 1·10⁴ to 5·10⁶g·mol⁻¹.
 5. The method according to claim 1, wherein the protic solventincludes water and aqueous solutions of salts of a general formula MX,where M is an alkali metal and X is selected from the group includingHCO₃ ⁻, CO₃ ²⁻, PO₄ ³⁻, HPO₄ ²⁻, H₂PO₄ ⁻ and halogens.
 6. The methodaccording to claim 5, wherein the concentration of the aqueous saltsolutions is 0 to 15% by weight.
 7. The method according to claim 1,wherein reaction of the hyaluronic acid, 4-R-TEMPO and the co-oxidant isperformed at the temperature within the range from −10 to 20° C. for 5minutes to 24 hours.
 8. The method according to claim 1, wherein theco-oxidant is present in an amount within the range from 0.05 to 1 molarequivalent with respect to the hyaluronic acid.
 9. The method accordingto claim 1, wherein the solvent is water or a mixture of water and apolar aprotic solvent with the content of water of at least 30% (v/v).10. The method of modification according to claim 1, wherein theoxidized hyaluronic acid derivative reacts with the amino acid.
 11. Themethod of modification according to claim 1, wherein the oxidizedhyaluronic acid derivative reacts with the peptide.
 12. The method ofmodification according to claim 1, wherein the oxidized hyaluronic acidderivative reacts with the polysaccharide containing a free amino group.13. The method of modification according to claim 1, wherein theoxidized hyaluronic acid derivative reacts with the amine, amino acid,peptide, or polysaccharide and the amount of the amine, amino acid,peptide or polysaccharide is within the range from 0.05 to 10 molarequivalents with respect to the hyaluronic acid derivative.
 14. Themethod of modification according to claim 1, wherein the oxidizedhyaluronic acid derivative reacts with the amine, amino acid, peptide,or polysaccharide and the reaction of the oxidized hyaluronic acidderivative with the amine, amino acid, peptide or polysaccharide isperformed in water or in a system of water-organic solvent at thetemperature within the range from 0 to 80° C. for 1 minute to 24 hours.15. The method of modification according to claim 14, wherein theoxidized hyaluronic acid derivative reacts with the amine, amino acid,peptide, or polysaccharide and the reaction of the oxidized hyaluronicacid derivative with the amine, amino acid, peptide or polysaccharide isperformed in the presence of NaBH₃CN as a reduction agent, which isadded to the reaction mixture in time 0 to 100 hours after the additionof the amine, amino acid, peptide or polysaccharide, to form an aminoform of HA of a general formula II


16. The method of modification according to claim 15, wherein the amountof NaBH₃CN as a reduction agent is within the range from 0 to 20 molarequivalents with respect to the molar amount of the aldehyde.
 17. Themethod of modification according to claim 14, wherein the organicsolvent is selected from the group comprising water-miscible alcohols,and water-miscible polar aprotic solvents.
 18. The method ofmodification according to claim 14, wherein the amount of water is atleast 50% (v/v) with respect to the volume of the whole solution.